Carrageenan-induced paw edema in the rat has been a standard in vivo model for the pharmacological evaluation of systemically administered anti-inflammatory agents. See Winter et al., Proc Soc Exp Biol Med 111: 544-547 (1962) and Vinegar et al., J Pharmacol Exp Ther 166: 96-103 (1969). In this model, subplantar administration of irritant into the hind paw of the rat causes a local inflammatory response which can be inhibited by steroidal and non-steroidal anti-inflammatory agents.
A related model has been developed by Williams et al., Curr Eye Res 2: 465-470 (1982) for the evaluation of topical ocular anti-inflammatory agents. In this model, bacterial endotoxin (LPS) is administered into the vitreous of the rabbit eye, resulting in an inflammation of the uvea characterized by accumulation of neutrophils (PMNs) in ocular tissue. These investigators utilized a biochemical method that allows indirect quantification of tissue-associated PMNs by assessing myeloperoxidase (MPO) activity contained in the azurophilic granules of the cell.
An alternative model of endotoxin-induced uveitis has been established in rats. (See, Forrester et al., Graefes Arch Klin Exp Ophthalmol 213: 221-233 (1980) and Bhattacherjee et al., Invest Ophthalmol Vis Sci 24: 196-202 (1983)). In this model, PMN influx into ocular tissue following subplantar or intravitreal injection of LPS is quantified by counting stained cells in a known volume of aqueous humor. See, also, Williams et al., Exp Eye Res 42: 211-218 (1986) and Tsuji et al., et al., Exp Eye Res 64: 31-36 (1997).
Each of the above approaches used to quantify ocular PMN content in models of endotoxin-induced uveitis has inherent shortcomings. Specifically, Bhattacherjee et al., Invest Ophthalmol Vis Sci 24: 196-202 (1983) reported considerable disparity in cell counts (as high as 40%) between the eyes of a single test animal when quantifying microscopically stained cells obtained by anterior chamber tap. This approach requires thorough mixing of the aqueous humor to dislodge tissue-adherent leukocytes prior to fluid removal in order to obtain a representative and homogeneous sample. Avidly adherent cells may not be dislodged and, therefore, not quantified by this procedure.
Similarly, the biochemical assessment of PMN-associated MPO activity employed by Williams et al., Curr Eye Res 2: 465-470 (1982) has notable deficiencies. Although endotoxin-induced uveitis in rabbits theoretically offers the advantage of providing a relatively large ocular globe, technical issues associated with tissue preparation and subsequent fractionation of large extract volumes make evaluation of total ocular PMN content cumbersome. This approach requires tissue extraction and solubilization of the MPO with hexadecyltrimethylammonium bromide (HTA-Br). See, Bradley et al., J Invest Dermatol 78: 206-209 (1982).
Although PMN-derived MPO primarily catalyzes hydrogen peroxide (H.sub.2 O.sub.2)-dependent oxidation of halides (i.e., chloride ions) physiologically, its ability to use artificial electron donors such as o-dianisidine (o-DA) as substrates has become the basis of the spectrophotometric determination of enzyme activity and a means of quantifying PMNs in inflamed tissue. Modifications of this method have been introduced to circumvent artifacts contributed by tissue/blood constituents, including cytosolic enzymes such as catalase, glutathione peroxidase, and heme proteins, and reducing substrates such as glutathione (GSH) and ascorbic acid (ASC). See, for example, Grisham et al., Methods in Enzymology, Vol. 186, Ed. Packer et al., San Diego: Academic Press, pp. 729-742 (1990), Liem et al., Anal Biochem 98: 388-393 (1979), and Egan et al., Agents Actions 29: 266-276 (1990).
Since the initial report of ocular PMN accumulation in the rabbit model of endotoxin-induced uveitis (Williams et al., Curr Eye Res 2: 465-470 (1982)), however, little consideration has been given to the presence of endogenous electron donors that may interfere in the assay of PMN-associated MPO activity. There is a need for a more reliable analytical method of quantifying PMN content of inflamed tissues, one that is not affected by endogenous tissue constituents.